Project Summary: Our long-term objective is to commercialize a fast, simple, flexible, and economical system for affinity-tag protein purification and tag removal. This technology was developed as a result of NIH-funded studies on the folding, stability and enzymology of the Bacillus protease subtilisin. The transformative purification technology combines three components: 1) a regulated, highly-specific protease (Psub) that is constitutively inactive; 2) a small molecule (imidazole) that activates the Psub; 3) a high-affinity inhibitor (Pro). Immobilized Psub (in the off-state) can capture a Pro-fusion protein from a cellular extract, allowing contaminants to be washed away. Subsequent addition of imidazole to the immobilized complex releases pure, tag-free target protein and leaves the Pro-tag tightly bound to Psub. Recombinant proteins are frequently fused with other proteins or peptides to facilitate expression and purification. The tags enable target protein binding in affinity purification, but ultimately must be processed by a site-specific protease. Tag removal, however, is frequently expensive, inefficient, and sometimes problematic. The technical innovation of our system is the integration of seamless tag removal into the purification process. This provides simplicity, efficiency, and robustness that is not available in any other system. Improved understanding of protein engineering principles has resulted in highly-efficient Psubs and Pro affinity tags. The experimental plan has two goals. The first is to generate economical materials for purification of standard proteins without tags. The second goal more ambitious goal is to develop methods for purifying high-value proteins that are either difficult or impossible with current methods. The Specific Aims are: 1) Evaluation of site-specific immobilization methods for Psub; 2) Cloning of challenging, high-value target proteins; 3) Expression-extraction-purification of high-value target proteins; 4) Optimization of the purification of challenging proteins; 5) Testing physical and biological properties of purified proteins. Better purification technology should advance in all areas of biology but have a particular impact on the production of growth factors needed for the growth, survival, and differentiation of therapeutic cells.